Project Summary ? Plasmodium vivax presents unique challenges to malaria elimination because it produces hypnozoites, dormant liver-stages that cause relapse infections from weeks to years without mosquito transmission. If untreated, hypnozoites represent a disease reservoir whose extent is unknown. Our studies in Madagascar provide evidence that P. vivax is now able to infect red blood cells of Duffy- negative people, demonstrating the capacity to evolve beyond a significant previously recognized barrier. At this time, there is also greater recognition of the clinical severity of vivax malaria and the capacity of this parasite to persist despite availability of bed nets and drugs that target blood stage parasites. To address this substantial public health challenge and threat to malaria elimination, efforts must focus on reducing the hypnozoite reservoir. Primaquine (PQ) is the only WHO-recommended drug that is able to kill hypnozoites and achieve radical cure of P. vivax. However, a number of factors must be considered regarding optimal use of this important antimalarial drug. Genetic variation in the gene encoding the human cytochrome P450 isoenzyme 2D6 (CYP2D6) has been associated with PQ failure through P. vivax relapses in people who have received standard PQ treatment (0.25-0.50 mg/kg body weight by mouth daily for 14 days). PQ may also cause life-threatening hemolytic anemia in G6PD deficient (G6PDd) people if drug treatment is not curtailed after signs of hemolysis become evident (usually hematuria). These observations emphasize the importance of developing effective strategies to use PQ and other 8-aminoquinoline drugs (tafenoquine; TQ). TQ delivered as a single-dose treatment (recently FDA-approved; not yet WHO-recommended) would improve adherence, but its much longer half-life (PQ ?5 hours; TQ ?15 days) makes it particularly dangerous in people with the most severe form of this enzymopathy. Here, we focus on the need to optimize PQ treatment. Our preliminary results reveal complex polymorphism in the CYP2D6 gene, significant variation in activity scores associated with the probe drug dextromethorphan (DM), and increasing variation in PQ effectiveness against P. vivax. We will address these challenges through the following Specific Aims. Aim 1: Evaluate CYP2D6 diplotypes and genome variation to identify individuals to be studied for identifying modifiers of PQ metabolism and develop a global framework to estimate PQ effectiveness. Aim 2: Evaluate PQ metabolism phenotype and CYP2D6 diplotype association in uninfected Malagasies. Aim 3: Assess PQ radical cure of P. vivax vs. recurrence in association with CYP2D6 and genomic variation in Malagasy study participants. Nearly 2.5 billion people are at risk of P. vivax malaria. Human genetic variation complicates population-based treatment using PQ in P. vivax-endemic areas. The proposed studies will investigate CYP2D6 genetic variation that confounds optimal use of PQ to develop population-based strategies to eliminate P. vivax.